Fast and simple purification of chemically modified hammerhead ribozymes using a lipophilic capture tag

A new type of 5′-lipophilic capture tag is described, enabling the facile reverse phase HPLC purification of chemically modified hammerhead ribozymes (oligozymes) whilst still carrying the 2′-O-tert.-butyldimethylsilyl protection of the essential riboses. In its most convenient form, the capture tag...

Full description

Saved in:

Bibliographic Details
Published in:	Nucleic acids research Vol. 27; no. 8; pp. 1950 - 1955
Main Authors:	Sproat, Brian S., Rupp, Thomas, Menhardt, Norbert, Keane, Doreen, Beijer, Barbro
Format:	Journal Article
Language:	English
Published:	England Oxford University Press 15-04-1999 Oxford Publishing Limited (England)
Subjects:	Cholesterol Chromatography, High Pressure Liquid - methods Electrophoresis, Capillary - methods Oligonucleotides - chemical synthesis Oligonucleotides - isolation & purification RNA, Catalytic - chemical synthesis RNA, Catalytic - isolation & purification Time Factors Vitamin E
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	A new type of 5′-lipophilic capture tag is described, enabling the facile reverse phase HPLC purification of chemically modified hammerhead ribozymes (oligozymes) whilst still carrying the 2′-O-tert.-butyldimethylsilyl protection of the essential riboses. In its most convenient form, the capture tag consists of a simple diol, such as hexan-1,6-diol, which at one end is attached via a silyl residue to a highly lipophilic entity such as tocopherol (vitamin E) or cholesterol, and the other end is functionalized as a phosphoramidite. This lipophilic capture tag is added as the last residue in the solid-phase synthesis of chemically modified hammerhead ribozymes. Cleavage from the support and release of all protecting groups except for the silyl groups is achieved with ethanolamine/ethanol. The crude product is then loaded directly on to a reverse phase HPLC column. Separation of failure peaks from full length product is achieved easily using a short run time. The retarded product peak is collected, lyophilized, desilylated in the normal way and then desalted. This method removes the lipophilic capture tag yet leaves behind the hexanediol entity which helps protect the compound against degradation by 5′-exonucleases. The purity of the product as judged by analytical anion-exchange HPLC and capillary gel electrophoresis is generally better than 95% full-length, and yields of 2–4 mg from a 1 µmol scale synthesis are routine. In addition, the method can be readily scaled up, an important feature for the development of such chemically modified ribozymes as potential therapeutics.
Bibliography:	istex:C316F63466D6B5CD33A29D80705C65BA084A0DAF ark:/67375/HXZ-39S0MMH9-3 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0305-1048 1362-4962
DOI:	10.1093/nar/27.8.1950